Novel Adenovirus Vectors

ABSTRACT

An adenoviral vector comprising a promoter further comprising a fragment of the 5′ untranslated region of the CMV IE1 gene including intron A and a nucleic acid sequence encoding a pathogen or tumour antigen for use as a medicament.

FIELD OF THE INVENTION

This invention relates to novel immunogenic adenovirus vectorcompositions and to their use in immunisation.

BACKGROUND

Vaccination has proved to be one of the most effective means ofpreventing diseases, particularly infectious diseases. Most vaccineswork by inducing antibodies that are protective against infection by therelevant pathogen. However many new vaccines target the cellular arm ofthe immune system and work by inducing effector and memory T cells.These can target intracellular pathogens and tumours. Many new T cellinducing vaccines that may be used either prophylactically ortherapeutically are in development.

T cells induced by vaccination may be useful in various ways. As well asreducing risk of diseases in the vaccinee they may be used in adoptivetransfer protocols to reduce risk of infection or disease in thosereceiving these cells. They may also be useful diagnostically.

An increasingly widely used method of inducing an immune response is toclone an antigen or epitope of interest into a vector. Vectors may beplasmid, bacterial or viral. Plasmid DNA vaccines are under intensivedevelopment and a variety of viral vectors appear useful forvaccination. These include poxviruses such as modified vaccinia virusAnkara (MVA), avipox vectors such as fowlpox and canarypox and ALVAC,herpesvirus vectors (including herpes simplex and CMV), alphaviruses andadenoviruses. There is increasing interest in the use of adenoviruses asvaccine vectors because of their ability to induce strong cellular andantibody responses.

Diseases that might be targeted by improved adenovirus vectors includebut are not limited to malaria, tuberculosis, HIV/AIDS, HCV, HBV, HSV,HPV, CMV, diseases caused by encapsulated bacteria such as thepneumococcus, parasitic diseases such as leishmaniasis, and a wide rangeof tumours and cancers, such as lymphoma, leukaemias, melanoma, renal,breast, lung, prostate, pancreatic and colorectal cancers.

SUMMARY OF THE INVENTION

The present invention is based on the inventors surprising discoverythat in adenoviral vector vaccines increasing the length of theheterologous promoter which controls expression of the antigen ofinterest enhances adenoviral vector immunogenicity and protectiveefficacy.

Adenoviruses form the family Adenoviridae and are classified into fivegenera (1). First isolated in 1953 from human adenoid tissue removedduring tonsillectomy (2), a vast number of species have now beendescribed that are infective to humans and a wide range of animals. Alladenoviruses have a similar virion—medium-sized (60-90 nm),non-enveloped, icosahedral particles, with a protein capsid (240 hexonsand 12 pentons) enclosing a ˜34-43 kbp double-stranded DNA genome withinthe core (3). Fifty-one human adenovirus (AdHu) serotypes have so farbeen described, based on serological studies of cross-neutralisingantibody responses to the hexon protein and terminal knob of the pentonfibre. These serotypes have been further grouped into six subgroups orspecies (A-F) within the Mastadenovirus genus, based on phylogeneticanalysis and their haemagglutination reaction (1). Adenoviruses show abroad tropism with most human serotypes, including the widely studiedAdHu5 (subgroup C), initially binding to the Coxsackie adenovirusreceptor (CAR) (4), followed by internalisation of the virion upon theinteraction of Arg-Gly-Asp (RGD) motifs in the penton base with α_(v)β₃-or α_(v)β₅-integrins (5). CAR is widely expressed on many cell types,but only on dendritic cells (DCs) at low levels. Some viruses withinsubgroup B do not bind CAR. AdHu35, for example, binds the complementregulatory protein membrane cofactor protein (MCP/CD46) (6), whilstAdHu3 attaches to the costimulatory molecules CD80 (B7.1) and CD86(B7.2) expressed by APCs (7). Some serotypes are ubiquitous and infectmost children during early infancy, such as AdHu1, 2 and 5, causingacute mild upper respiratory infections. Others, however, can lead toserious and even fatal infections, such as pneumonia (AdHu3 and 7),especially in immunocompromised individuals (8) and children (9).

Adenoviruses were initially developed as vehicles for gene therapy.Attempts to replace missing or faulty genes by adenoviral gene transferwere largely unsuccessful in experimental animals and human volunteersalike due to innate and adaptive immune responses induced by theadenoviral antigens (3). However, the demonstration by gene therapistsof the induction of potent cellular and humoral transgene-specificimmune responses pioneered the use of these viruses as vaccine vectorswith highly successful results first demonstrated using a recombinantrabies virus glycoprotein (10). The adenoviral genome is wellcharacterised and comparatively easy to manipulate (11, 12). Deletion ofcrucial regions of the viral genome, such as E1, renders the vectorsreplication-defective, which increases their predictability andeliminates unwanted pathogenic side effects. Replication-deficientadenoviruses can be grown to high titre in tissue culture, using celllines that provide the missing essential E1 gene products in trans (13).They can be applied systemically as well as through mucosal surfaces andtheir relative thermostability facilitates their clinical use. Whilstbovine, porcine, and ovine adenoviruses are being explored forveterinary use (3), studies of adenovirus vectors of differing humanserotype have shown variable immunogenicity. The majority of studies nowfocus on the most promising candidates, including AdHu5, AdHu35 andAdHu11. These vectors can induce potent and protective T and Bcell-mediated responses against a range of viral and parasitic encodedantigens (10, 14-17). However, problems surrounding pre-existingimmunity to ubiquitous viruses such as AdHu5 and AdHu35 remain a bighurdle to the clinical deployment of these vectors. Depending on theregion under study, 35-80% of human adults carry AdHu5-neutralisingantibodies, and 5-15% AdHu35-neutralising antibodies (18).

E1-deleted replication-defective adenovirus vectors can be generatedfrom “molecular clones”, in which the entire genome is carried within abacterial plasmid (11). Vaccine constructs can be ligated into theE1-deletion site using commercially available kits. Upon removal of thebacterial sequences by restriction enzyme digest, and exposure of theinverted terminal repeats (ITRs), the plasmid can be transfected into apackaging cell line that supplies the essential E1 gene product intrans, thus generating the pure recombinant virus.

The adenoviral capsid will only allow a 5% increase in genome sizebefore efficient packaging and viral stability is disrupted—an extra 1.8kbp in the case of the well-studied vector AdHu5 (3). Vectors deleted ofE1 and the non-essential E3 region (21) can accommodate up to 7.5 kbp offoreign DNA and remain the leading choice for vaccine studies using thisvector.

Therefore, according to a first aspect of the present invention there isprovided an immunogenic composition comprising an adenoviral vector,said adenoviral vector further comprising a promoter comprising afragment of the 5′ untranslated region of the CMV IE1 gene includingintron A and a nucleic acid sequence encoding a pathogen or tumourantigen under the control of said promoter; wherein said antigen is nota murine malaria parasite antigen.

In one embodiment, the composition may be a vaccine composition.Preferably, the vaccine composition is suitable for human administrationand can be used to elicit a protective immune response against theencoded antigen.

In a preferred embodiment, the adenoviral vector is a simian adenoviralvector. More preferably, the simian adenoviral

In a preferred embodiment, the adenoviral vector is a simian adenoviralvector. More preferably, the simian adenoviral vector is AdC6 (C6), AdC7(C7), AdC9 (C9) vector. These viruses are detailed by S. Roy et al.Virology (2004) Volume 324, pp 361-372. Therein AdC6 is referred to asSAdV-23; AdC7 is referred to as SAdV-24; and AdC9 is referred to asSAdV-25. In other publications AdC9 is also called AdC68 (e.g.Fitzgerald et al. J Immunology 2003, 170:1416-22).

It will be understood that the development of simian adenovirus vectors,for example, chimpanzee adenoviruses, against which pre-existingimmunity is prevalent neither in humans (1-2%) nor in some other simianspecies, such as rhesus macaques, often used for pre-clinical testing(19, 20) is desirable.

It will be further understood that in many applications it is preferablefor the adenovirus vector to be replication deficient meaning that theyhave been rendered incapable of replication because of a functionaldeletion, or complete removal, of a gene encoding a gene productessential for viral replication. By way of example, the vectors of theinvention may be rendered replication defective by removal of all or apart of the E1 gene, and optionally also the E3 region and/or the E4region.

It should be understood that CMV promoters are well known in the art.Numerous versions of the CMV Immediate Early (IE) promoter exist asshown in FIG. 1. It is known that these can be used to drive antigenexpression in host eukaryotic cells (22). The CMV IE enhancer-promoterhas been shown to cause high levels of transgene expression ineukaryotic tissues when compared with other promoters. A DNA vaccineexpressing the HIV-1 antigens Gag/Env under the control of the CMVpromoter, rather than the endogenous AKV murine leukaemia virus longterminal repeat, was shown to be more immunogenic in macaques (23).

It is further known that inclusion of the CMV intron A results inenhanced transgene expression over the CMV IE enhancer-promoter alone invitro and in vivo (24, 25) using plasmid DNA vectors.

However, no assessments of the comparative immunogenicity of thesevectors with different promoters has been undertaken.

More recently expression of a firefly luciferase gene has been assessedusing an AdHu5 vector and better expression observed with the additionof the intron A sequence (26). Again no studies of immune responses wereundertaken. It has been suggested that inclusion of the intron mayenhance the rate of polyadenylation and/or nuclear transport associatedwith splicing of pre-mRNA primary transcripts (27). Such research onpromoter function with adenovirus and plasmid vectors has been directedat enhancing transgene expression in order to improve the efficacy ofgene therapy vectors, where the desired outcome is high level prolongedexpression of the transgene.

It will be apparent to the skilled person that, although some expressionis required for immunogenicity, increased expression of a gene does notcorrelate with increased immunogenicity. Indeed increased expression ofa transgene may lead to vector instability or non-viability of therecombinant virus.

It will be apparent that the antigen can be any antigen of interesteither exogenous or endogenous. Exogenous antigens include all moleculesfound in infectious organisms. For example bacterial immunogens,parasitic immunogens and viral immunogens.

Bacterial sources of these immunogens include those responsible forbacterial pneumonia, meningitis, cholera, diphtheria, pertussis,tetanus, tuberculosis and leprosy.

Parasitic sources include malarial parasites, such as Plasmodium, aswell as trypanosomal and leishmania species.

Viral sources include poxviruses, e.g., smallpox virus, cowpox virus andorf virus; herpes viruses, e.g., herpes simplex virus type 1 and 2,B-virus, varicella zoster virus, cytomegalovirus, and Epstein-Barrvirus; adenoviruses, e.g., mastadenovirus; papovaviruses, e.g.,papillomaviruses such as HPV16, and polyomaviruses such as BK and JCvirus; parvoviruses, e.g., adeno-associated virus; reoviruses, e.g.,reoviruses 1, 2 and 3; orbiviruses, e.g., Colorado tick fever;rotaviruses, e.g., human rotaviruses; alphaviruses, e.g., Easternencephalitis virus and Venezuelan encephalitis virus; rubiviruses, e.g.,rubella; flaviviruses, e.g., yellow fever virus, Dengue fever viruses,Japanese encephalitis virus, Tick-borne encephalitis virus and hepatitisC virus; coronaviruses, e.g., human coronaviruses; paramyxoviruses,e.g., parainfluenza 1, 2, 3 and 4 and mumps; morbilliviruses, e.g.,measles virus; pneumovirus, e.g., respiratory syncytial virus;vesiculoviruses, e.g., vesicular stomatitis virus; lyssaviruses, e.g.,rabies virus; orthomyxoviruses, e.g., influenza A and B; bunyavirusese.g., LaCrosse virus; phieboviruses, e.g., Rift Valley fever virus;nairoviruses, e.g., Congo hemorrhagic fever virus; hepadnaviridae, e.g.,hepatitis B; arenaviruses, e.g., 1cm virus, Lasso virus and Junin virus;retroviruses, e.g., HTLV I, HTLV II, HIV-1 and HIV-2; enteroviruses,e.g., polio virus 1, 2 and 3, coxsackie viruses, echoviruses, humanenteroviruses, hepatitis A virus, hepatitis E virus, and Norwalk-virus;rhinoviruses e.g., human rhinovirus; and filoviridae, e.g., Marburg(disease) virus and Ebola virus.

Antigens from these bacterial, viral and parasitic sources can beconsidered as exogenous antigens because they are not normally presentin the host and are not encoded in the host genome.

In contrast, endogenous antigens are normally present in the host or areencoded in the host genome, or both. The ability to generate an immuneresponse to an endogenous antigen is useful in treating tumours thatbear that antigen, or in neutralising growth factors for the tumour. Anexample of the first type of endogenous antigen is HER2, the target forthe monoclonal antibody called Herceptin. An example of the second,growth factor, type of endogenous antigen is gonadotrophin releasinghormone (called GnRH) which has a trophic effect on some carcinomas ofthe prostate gland.

Preferably, the antigen is an antigen from an infectious pathogen ofhumans or livestock.

In one preferred embodiment, the antigen is from a pathogen which causesmalaria. Preferably, the antigen is a P. falciparum antigen.

Preferably, the malaria antigen is a pre-erythrocytic or blood-stagemalaria antigen.

In particularly preferred embodiments of the present invention, themalaria antigen is ME-TRAP, CSP, MSP-1 or fragments thereof, or AMA1.

Preferably, when the malaria antigen is an MSP-1 antigen it has thesequence of PfM117 (SEQ ID NO. 1) or PfM128 (SEQ ID NO. 3).

Malaria is a disease against which it has been very difficult togenerate protective immunity in both humans and small animal models.Thus the results discussed below indicate that the use of theimmunisation approaches described herein have general potential for usein generating very potent vaccines in humans and other species.

In a further preferred embodiment, the antigen in a mycobacterialantigen. Preferably, the antigen is a M. tuberculosis antigen. Morepreferably, the antigen is M. tuberculosis antigen 85A.

The inventors have found that the enhanced antigen expression resultingfrom the presence of the long CMV promoter including intron A leads to aremarkably large and surprising increase in the immunogenic potency ofthese vaccine vectors, and to enhanced protective efficacy againstpathogen challenge.

The above immunogenic viral vector compositions, may be formulated intopharmaceutical dosage forms, together with suitable pharmaceuticallyacceptable carriers, such as diluents, fillers, salts, buffers,stabilizers, solubilizers, etc. The dosage form may contain otherpharmaceutically acceptable excipients for modifying conditions such aspH, osmolarity, taste, viscosity, sterility, lipophilicity, solubilityetc.

Suitable dosage forms include solid dosage forms, for example, tablets,capsules, powders, dispersible granules, cachets and suppositories,including sustained release and delayed release formulations. Powdersand tablets will generally comprise from about 5% to about 70% activeingredient. Suitable solid carriers and excipients are generally knownin the art and include, e.g. magnesium carbonate, magnesium stearate,talc, sugar, lactose, etc. Tablets, powders, cachets and capsules areall suitable dosage forms for oral administration.

Liquid dosage forms include solutions, suspensions and emulsions. Liquidform preparations may be administered by intravenous, intracerebral,intraperitoneal, intradermal, parenteral or intramuscular injection orinfusion. Sterile injectable formulations may comprise a sterilesolution or suspension of the active agent in a non-toxic,pharmaceutically acceptable diluent or solvent. Suitable diluents andsolvents include sterile water, Ringer's solution and isotonic sodiumchloride solution, etc. Liquid dosage forms also include solutions orsprays for intranasal administration.

Aerosol preparations suitable for inhalation may include solutions andsolids in powder form, which may be combined with a pharmaceuticallyacceptable carrier, such as an inert compressed gas.

Also encompassed are dosage forms for transdermal administration,including creams, lotions, aerosols and/or emulsions. These dosage formsmay be included in transdermal patches of the matrix or reservoir type,which are generally known in the art.

Pharmaceutical preparations may be conveniently prepared in unit dosageform, according to standard procedures of pharmaceutical formulation.The quantity of active compound per unit dose may be varied according tothe nature of the active compound and the intended dosage regime.

The active agents are to be administered to human subjects in“therapeutically effective amounts”, which is taken to mean a dosagesufficient to provide a medically desirable result in the patient. Theexact dosage and frequency of administration of a therapeuticallyeffective amount of active agent will vary, depending on such factors asthe nature of the active substance, the dosage form and route ofadministration.

The medicaments and pharmaceutical compositions of the present inventionmay be administered systemically or locally. This is applicable to boththe use and method aspects of the invention equally. Systemicadministration may be by any form of systemic administration known, forexample, orally, intravenously or intraperitoneally. Localadministration may be by any form of local administration known, forexample topically.

In particularly preferred embodiments the pharmaceutical compositionincludes at least one pharmaceutically acceptable excipient.

According to a second aspect of the present invention there is providedan adenoviral vector comprising a promoter further comprising a fragmentof the 5’ untranslated region of the CMV IE1 gene including intron A anda nucleic acid sequence encoding a pathogen or tumour antigen under thecontrol of said promoter for use as a medicament.

Preferably, the adenoviral vector is a simian adenoviral vector.

Preferably the adenoviral vector is replication deficient.

Preferably, the antigen is an antigen from an infectious pathogen ofhumans or livestock.

According to a third aspect of the present invention there is providedthe use of an adenoviral vector comprising a promoter further comprisinga fragment of the 5′ untranslated region of the CMV IE1 gene includingintron A and a nucleic acid encoding a malarial antigen in themanufacture of a vaccine or immunotherapeutic for the prevention ortreatment of malaria.

Preferably, the malaria antigen is not from a murine parasite.

Preferably, the adenoviral vector is a simian adenoviral vector.

Preferably the adenoviral vector is replication deficient.

In a preferred embodiment, the encoded malaria antigen is a P.falciparum antigen. More preferably, the malarial antigen is apre-erythrocytic or blood-stage malaria antigen. Even more preferably,the malarial antigen is ME-TRAP, CSP, MSP-1 or fragments thereof, orAMAl.

In a most preferred embodiment, when the malarial antigen is an MSP-1antigen it has the sequence of PfM117 (SEQ ID NO. 1) or PfM128 (SEQ IDNO. 3).

According to a fourth aspect of the present invention there is providedthe use of an adenoviral vector comprising a promoter further comprisinga fragment of the 5′ untranslated region of the CMV IE1 gene includingintron A and a nucleic acid encoding a M. tuberculosis antigen in themanufacture of a vaccine or immunotherapeutic for the prevention ortreatment of tuberculosis.

Preferably, the adenoviral vector is a simian adenoviral vector.

Preferably the adenoviral vector is replication deficient.

In a preferred embodiment, the encoded antigen is the M. tuberculosisantigen 85A.

According to a fifth aspect of the present invention there is providedan adenoviral vector comprising a promoter further comprising a fragmentof the 5′ untranslated region of the CMV IE1 gene including intron A anda nucleic acid encoding a malarial antigen for use in the prevention ortreatment of malaria.

Preferably, the malaria antigen is not from a murine parasite.

Preferably, the adenoviral vector is a simian adenoviral vector.

Preferably the adenoviral vector is replication deficient.

Preferably, the malaria antigen is a P. falciparum antigen.

Preferably, the malaria antigen is a pre-erythrocytic or blood-stagemalaria antigen.

Preferably, the malaria antigen is ME-TRAP, CSP, MSP-1 or fragmentsthereof, or AMA1. More preferably, the malaria antigen is an MSP-1antigen has the sequence of PfM117 (SEQ ID NO. 1) or PfM128 (SEQ ID NO.3).

According to a sixth aspect of the present invention, there is providedan adenoviral vector comprising a promoter further comprising a fragmentof the 5′ untranslated region of the CMV IE1 gene including intron A anda nucleic acid encoding a M. tuberculosis antigen for use in theprevention or treatment of tuberculosis.

Preferably, the adenoviral vector is a simian adenoviral vector.

Preferably, the adenoviral vector is replication deficient.

In a preferred embodiment, the encoded antigen is M. tuberculosisantigen 85A.

It will be readily apparent that the medicaments described in any of theabove aspects may comprise one or more pharmaceutically acceptablevehicles, carriers, diluents, excipients or adjuvants.

According to a seventh aspect of the present invention there is provideda product, combination or kit comprising;

a) a priming composition comprising an adenoviral vector, saidadenoviral vector further comprising a long heterologous promoter,wherein the promoter is a fragment of the 5′ untranslated region of theCMV IE1 gene including intron A, and at least one nucleic acid sequenceencoding a pathogen or tumour antigen, wherein the antigen is not amurine malaria parasite antigen; and

b) a boosting composition comprising a recombinant pox virus vector,said pox virus vector further comprising at least one nucleic acidsequence encoding a pathogen or tumour antigen which is the same as atleast one antigen of the priming composition.

Preferably, the adenoviral vector is a simian adenoviral vector. Morepreferably, the simian adenoviral vector is AdC6 (C6), AdC7 (C7), orAdC9 (C9) vector.

Preferably, the antigen is not a murine malaria parasite antigen.

In a preferred embodiment, the promoter excludes Exon B.

Preferably, the antigen is an antigen from an infectious pathogen ofhumans or livestock.

In one preferred embodiment, the antigen is from a pathogen which causesmalaria. Preferably, the antigen is a P. falciparum antigen. Morepreferably, a pre-erythrocytic or blood-stage malarial antigen. Evenmore preferably, the malarial antigen is ME-TRAP, CSP, MSP-1 orfragments thereof, or AMA1.

When the malarial antigen is an MSP-1 antigen preferably it has thesequence of PfM117 (SEQ ID NO. 1) or PfM128 (SEQ ID NO. 3).

In a further preferred embodiment, the antigen in a mycobacterialantigen. More preferably, the antigen is a M. tuberculosis antigen. Evenmore preferably, the antigen is M. tuberculosis antigen 85A.

Also provided is the use of the combination for production of a kit forgenerating a protective T cell response against at least one targetantigen of a pathogen or tumour in a subject.

According to an eighth aspect of the present invention there is provideda method of eliciting an immune response in a subject comprisingadministering an effective amount of an immunogenic composition orvaccine according to the first aspect of the present inventionsufficient to elicit an immune response.

It will be apparent that the subject can be administered the compositionor vaccine for either prophylactic or immunotherapeutic purposes,depending on the antigen.

In a preferred embodiment, the subject is immunised using a heterologousprime-boost regimen.

The skilled person will understand that heterologous prime-boost refersto a regimen wherein an effective amount of a first immunogeniccomposition or vaccine according to the present invention isadministered to an individual at a first time point and subsequently aneffective amount of a second immunogenic composition or vaccine encodingthe same antigen as the immunogenic composition or vaccine according tothe present invention is administered at a second time point. It will beunderstood that in an heterologous prime-boost regimen the first andsecond immunogenic composition or vaccines are different.

Preferably, the second immunogenic composition or vaccine isadministered 2-8 weeks after the first immunogenic composition orvaccine.

It will be readily apparent to the skilled person that the term subjectas used in the present invention relates to any animal subject. This mayparticularly be a mammalian subject, including a human.

Thus products of the invention may be useful not only in human use butalso in veterinary uses, for example in the treatment of domesticatedmammals including livestock (e.g. cattle, sheep, pigs, goats, horses orin the treatment of wild mammals, such as those captive in zoos).

In another aspect, the product of the invention may be used for thetreatment of non-mammalian subjects, including fowl such as chickens,turkeys, duck, geese and the like.

According to a ninth aspect of the present invention there is provided asimian adenoviral vector comprising a long heterologous promoter,wherein the promoter is a fragment of the 5′ untranslated region of theCMV IE1 gene including intron A and at least one nucleic acid sequenceencoding a pathogen or tumour antigen of interest.

Preferably, the promoter does not include exon B.

Preferably the simian adenoviral vector is replication deficient.

Preferably, the antigen is an antigen from an infectious pathogen ofhumans or livestock.

In one preferred embodiment, the antigen is from a pathogen which causesmalaria. Preferably, the antigen is a P. falciparum antigen. Morepreferably, a blood-stage malarial antigen. Even more preferably, themalarial antigen is ME-TRAP, CSP, MSP-1 or fragments thereof, or AMA1.

When the malarial antigen is an MSP-1 antigen preferably it has thesequence of PfM117 (SEQ ID NO. 1) or PfM128 (SEQ ID NO. 3).

In a further preferred embodiment, the antigen in a mycobacterialantigen. More preferably, the antigen is a M. tuberculosis antigen. Evenmore preferably, the antigen is M. tuberculosis antigen 85A.

According to a tenth aspect of the present invention there is provided amethod for enhancing the T cell immunogenicity of an immunogenicadenoviral vector composition or vaccine according to the first aspect,comprising administering said vaccine in combination with a CpGadjuvant.

It will be apparent that the CpG adjuvant can be administered prior to,concomitantly with, or subsequently to said immunogenic composition orvaccine.

According to an eleventh aspect of the present invention there isprovided a composition comprising an immunogenic adenoviral vectorcomposition or vaccine according to first aspect and a CpG adjuvant.

According to a twelfth aspect of the present invention there is providedthe composition according to the eleventh aspect for use as amedicament.

According to a thirteenth aspect there is provided a kit comprising theimmunogenic adenoviral vector composition or vaccine of first aspect anda CpG adjuvant for use in generating an immune response in a subjectagainst at least one pathogen or tumour antigen.

It will be understood that an immunogenic composition referred to in anyof the above aspects may in certain embodiments be a vaccine.

It will be apparent that the antigen according to any aspect of thepresent invention may be any antigen of interest as described inrelation to the first aspect.

It will be apparent that any feature described as preferred inconnection with one aspect of the invention is also preferred inrelation to other aspects of the invention unless otherwise stated, andthat preferred embodiments relating to one feature are disclosed incombination with preferred embodiments relating to other features.

The invention will now be further described with reference to thefollowing examples and figure in which:

FIG. 1 shows CMV Promoters.

1) Complete CMV IE promoter sequence from GenBank. 2) The “long” 1.9 kbpversion of the promoter referred in this document. 3) Promoter withchimeric intron from Promega (Southampton, UK) used to express ME-TRAP.4) The “small” 0.6 kbp version of the promoter referred to in thisdocument.

FIG. 2 shows quantification of antigen expression by quantitativereal-time RT-PCR.

293A cells were infected with AdHu5 expressing (a) MSP-1₄₂ or (b) 85A,under the control of the long or small promoter. Cells were harvestedinto RLT buffer, and the RNA extracted and reverse transcribed intocDNA. The levels of MSP-1₄₂, 85A and AdHu5 E4orf1 cDNA target sequenceswere measured by real-time PCR. Relative gene expression was calculatedas the ratio of target antigen mRNA copies to E4orf1 copies. Each columnrepresents the mean ratio±S.E.

FIG. 3 shows quantification of MSP-1₄₂ antigen expression by WesternBlot.

293A cells were infected with no virus (lane 1) Ad42SP (lane 2) orAd42LP (lane 3) in cell culture medium excluding FCS. Cell culturesupernatants were harvested once 100% CPE was evident, and concentratedby centrifugation through Centricon YM 30 tubes (Millipore, Watford,UK). Proteins from cell culture supernatants were separated by SDS-PAGEand electroblotted onto nitrocellulose membrane, before staining withHRP-conjugated mAb to the C-terminal PK/V5 tag. The blot was developedand exposed to photographic film.

The predicted molecular mass for MSP-1₄₂-PK is 46 kDa.

FIG. 4. shows peptide-specific IFN-γ-secreting T cell responses inducedby (a) Ad-MSP-1₄₂ or (b) Ad-85A vaccination.

BALB/c mice were immunised i.d. with 10¹⁰ vp of each adenovirus, andresponses measured in the spleens of immunised mice 14 dayspost-immunisation by ex-vivo IFN-γ ELISPOT. Columns represent the meannumber of IFN-γ SFC per million splenocytes±S.E. (n=3 mice/group). *p≦0.05, ** p≦0.01, comparing responses between groups that wereimmunised with AdHu5 vectors expressing the relevant antigen under thecontrol of the long (LP) or short (SP) promoter.

FIG. 5. shows MSP-1₁₉-specific whole IgG antibody responses induced byAd42SP or Ad42LP.

BALB/c mice were immunised i.d. with 10¹¹ vp Ad42SP or with 5×10¹⁰ vpAd42LP. Whole IgG responses against MSP-1₁₉ were measured byanti-GST-MSP-1₁₉ ELISA in the serum of mice 13 days post-immunisation.GST controls all negative (data not shown). Columns represent the meanlog10 endpoint titre±95% C.I. (n=3 mice/group). * p<0.05, comparingresponses between groups.

FIG. 6. shows kinetics of MSP-1₁₉-specific whole IgG antibody responsesinduced by Ad42LP.

BALB/c mice were immunised once i.d. with 5×10¹⁰ vp Ad42 at week 0.Whole IgG responses against MSP-1₁₉ were assayed by anti-GST-MSP-1₁₉ELISA in the serum of mice taken at 14 day intervals. Points representthe results of two experiments as the mean log10 endpoint titre±95% C.I.(n=18 mice). *** p<0.001, comparing differences between time points bypaired analysis of data from individual mice.

FIG. 7. shows MSP-1₁₉-specific IgG antibody responses induced by AdM42prime-boost vaccination.

BALB/c mice were immunised i.d. with 5×10¹⁰ vp Ad42LP and boosted i.d.with 5×10⁷ pfu MVA expressing the same antigen either two or eight weekslater. Whole IgG responses against MSP-1₁₉ were measured byanti-GST-MSP-1₁₉ ELISA in the serum of mice 13 days after the secondimmunisation. Columns represent the results of two or three experimentsas the mean log10 endpoint titre±95% C.I. (n=11-22 mice/group). ***p≦0.001, comparing responses between the two groups.

FIG. 8. shows P. yoelii sporozoite challenge of AdM42 (8 wks) immunisedBALB/c mice.

BALB/c mice were immunised as described in table 1, and challenged with50 P. yoelii sporozoites 14 days after the final immunisation (day 0).Blood-stage parasitaemia was monitored daily by Giemsa-stainedthin-blood smear from day 5, and percentage pRBCs calculated. Resultsare shown for: (a) unimmunised naïve controls n=6; (b) AdM42 (8 wks)n=6.

Unprotected mice which succumbed to infection or were sacrificed (at 80%blood-stage parasitaemia) are indicated by the cross symbol t.

FIG. 9 shows immunogenicity to ME.TRAP.

(a) Breadth of the immune response to ME.TRAP. BALB/c mice wereimmunized with 1×10⁹ vp of adenoviral vectors coding for ME.TRAP. Immuneresponses were measured 2 weeks later by ELISPOT after stimulation ofcells with overlapping peptides covering the whole sequence of theME.TRAP transgene. Data are mean±s.d. for three mice per group. (b)Kinetics of the immune response to ME.TRAP. BALB/c mice were immunizedwith adenoviral (1×10¹⁰ vp) and poxviral vectors (1×10⁷ pfu). Themagnitude of the immune response was measured after stimulation ofsplenocytes with Pb9 peptide and detection of IFNgamma⁺-producing CD8⁺ Tcells by flow cytometry at different intervals. (c) Total number ofIFNgamma⁺ CD8⁺ T cells per spleen during the peak of the effector andmemory responses for each vector. Calculations were performed in thesame groups of mice from FIG. 1 b. (d) The percent of IFNgamma⁺ CD8⁺ Tcells from representative mice upon Pb9 peptide stimulation. Upper panelshows the peak of the effector response for each vector (20 dayspost-prime for adenoviral vectors and 7 days post-prime for poxviralvectors) The memory phase was measured at day 60 post-prime. Data aremean±s.e.m. for three mice per group.

FIG. 10 shows immunogenicity to ME.TRAP in C57BL/6 mice.

(a) Breadth of the immune response. Mice were immunized with 1×10⁹ vp ofadenoviral vectors coding for ME.TRAP. Immune responses were measured 2weeks later by ELISPOT after stimulation of cells with overlappingpeptides covering the whole sequence of the ME.TRAP transgene. Data aremean±s.d. for three mice per group. (b) The percent of IFNgamma⁺ CD8⁺and CD4⁺ T cells from a pool of 3 mice upon peptide stimulation. Upperpanel shows the CD8⁺ T-cell response for each vector (20 dayspost-prime) and lower panel shows the CD4+ T-cell response.

FIG. 11. shows acquisition of effector phenotype and cytolytic functionsby CD8⁺ T cells at different intervals post-vaccination.

BALB/c mice were immunized as described in FIG. 1. Data show percentageof CD8⁺ IFNgamma⁺ CD43^(hi) (a) and CD8⁺ IFNgamma⁺ Granzyme Bcoexpression (b). (c) Granzyme B expression from representative mice atindicated days post-prime. Histogram shows GrB expression (whitebackground) after staining with anti-human GrB, compared to an isotypecontrol (gray background). The number corresponds to MFI of the positivesample (black solid line). Data in graphs are mean±s.e.m. for three miceper group.

FIG. 12. shows analysis of the memory response by phenotypic markers.

BALB/c mice were immunized as described in FIG. 1. Splenocytes wereco-stained for CD8, IFNgamma⁺ and (a) CD62L, (b) CD127, (c) IL-2 and (d)CD27. Bars show percentages of cells within the IFNgamma⁺ compartment.Data are mean±s.e.m. for three mice per group.

FIG. 13. shows analysis of the antibody responses to Pf TRAP.

IgG antibodies against the TRAP region were analyzed by ELISA in serumfrom groups of at least 3 BALB/c mice after 2 weeks of immunization withindividual vectors. Results were reported as a dilution factor neededfor a sample in order to reach the O.D. of a naïve serum.

FIG. 14. shows immunogenicity of various adenoviruses encoding thePfM115 insert in BALB/c mice. After a single immunisation intradermally(5×10¹⁰ vp) with the various adenoviral vectors at week 0 good antibodylevels were detected to the 19 Kd fragment of PfMSP1 at 2 weeks in allmice and these titres increased up to week 8 when all mice wereadministered an MVA encoding the same insert, leading to an furtherincrease in antibody titres. The simian adenoviral vectors appearssimilar in immunogenicty to the AdHu5 vector. The AdHu5PfM115C4bpencodes an additional C-terminal core sequence from the complementprotein C4bp.

FIG. 15 shows assessment of the potential immune enhancing effect of aCpG sequence (CpG 1826) added to the AdHu5 PfM115 adenovirus vector.

BALB/c mice were immunised intradermally on one occasion and T cellresponses evaluated 14 days later. A large pool of overlapping peptidesspanning the insert were used to evaluate CD8 (above) and CD4 (below) Tcell IFN-gamma responses.

EXAMPLES

In the following Examples a number of antigens have been used in theadenovirus vector vaccines of the current invention:

The Mycobacterium tuberculosis antigen 85A (28, 29).

The 42 kDa C-terminus of the blood-stage malarial antigen merozoitesurface protein-1 (MSP-1₄₂) from the murine parasite Plasmodium yoelii(30).

The malaria sporozoite antigen circumsporozoite protein (CSP) from themurine malaria parasite P. berghei

The pre-erythrocytic malarial antigen insert multi-epitopestring—thrombospondin-related adhesion protein (ME-TRAP) from P.falciparum (31, 32).

A fusion protein of regions of the P. falciparum blood-stage antigenMSP-1 denoted PfM117

A fusion protein of regions of the P. falciparum blood-stage antigenMSP-1 denoted PfM128

Example 1 Production of Adenovirus Vector Vaccines Containing MurineMalaria Antigens and a Tuberculosis Antigen

1.1 Enhancement of Antigen Expression by CMV Promoter in RecombinantAdHu5 Vectors.

AdHu5 vectors encoding murine malaria P. yoelii MSP-1₄₂ or antigen85Afrom M. tuberculosis were compared, using vectors which drive transgeneexpression by either the “small” 0.6 kbp version of the CMV IE promoter(lacking intron A), or the “long” 1.9 kbp version of the promoter (withregulatory element, enhancer and intron A). The small and long versionsof the promoter are referred to as SP and LP respectively. The level ofantigen expression by AdHu5 vectors was assayed in vitro by quantitativereal-time RT-PCR (FIG. 2). The level of antigen expression wasnormalised to the AdHu5 E4orf1 transcript. In both cases, significantlyhigher levels of antigen expression were measured following infection of293A cells with AdHu5 vectors expressing antigen under the control ofthe long promoter. The overall level of antigen expression may beantigen dependent, given both vectors encoding 85A expressedsignificantly higher levels of antigen compared to either vectorencoding MSP-1₄₂. These results were confirmed for the vectors encodingMSP-1₄₂ by Western Blot (FIG. 3). The MSP-1₄₂ antigen includes the PKepitope (amino acid sequence IPNPLLGLD) as a C-terminal fusion. Antigenis detected using the monoclonal antibody anti-PK (also known asanti-V5) from Serotec (Oxford, UK).

1.2 Enhancement of T Cell Immunogenicity by CMV Promoters in RecombinantAdHu5 Vectors.

Groups of BALB/c mice were immunised intradermally (i.d.) with 10¹⁰ vpof each adenovirus, and responses measured in the spleen to known CD8⁺and CD4⁺ T cell epitopes 14 days later by ex-vivo interferon-gamma(IFN-γ) ELISPOT (FIG. 4). The epitopes in MSP-1₄₂ are all known H-2^(d)class I-restricted epitopes (FIG. 4 a). pll is a known H-2^(d) classI-restricted epitope in 85A, whilst p15 is class II-restricted (FIG. 4b). Responses were only detected against known epitopes in MSP-1₄₂ whenmice were immunised with Ad42LP, whereas responses to 85A were inducedby both vectors, with those against p15 tending to be stronger in theAd85ALP group. These data correlate with the level of antigen expressionmeasured by real-time RT-PCR (FIG. 2).

1.3 Enhancement of Antibody Immunogenicity by CMV Promoters inRecombinant AdHu5 Vectors.

Groups of BALB/c mice were immunised i.d. with 10¹⁰ vp of Ad42LP orAd42SP. Whole IgG antibody responses against the C-terminus of MSP-1₄₂(MSP-1₁₉) were assayed by ELISA two weeks later (FIG. 5). There was nodetectable antibody responses against MSP-1₁₉ following Ad42SPimmunisation, whereas Ad42LP primed a significantly higher response,with an endpoint titre of approximately 1000. This response, induced byAd42LP, continues to increase over time, reaching a plateau by 6-8 weeks(FIG. 6). This antibody response can be boosted to a significantlyhigher level by MVA encoding the same antigen. Antibody responses aresignificantly higher following this heterologous AdM prime-boost regime,if Ad42LP primed mice are boosted 8 weeks rather than 2 weeks later(FIG. 7).

1.4 Protection of Mice Against Lethal Blood-Stage P. Yoelii Challenge byAdM-MSP-1₄₂ Immunisation.

The protection provided by the prime boost regime was investigated byexamination of the protection provided by AdM-MSP-1₄₂ immunisationagainst lethal blood-stage P. yoelii challenge in mice. Groups of BALB/cmice were immunised i.d. with 5×10¹⁰ vp of Ad42LP and boosted with MVAexpressing the same antigen two or eight weeks later. All immunisationregimes utilised the Ad42LP vector, and MVA expressing the same antigen.Mice were challenged i.v. with 10⁴ P. yoelii pRBCs 14 days after thefinal immunisation. Homologous prime-boost regimes were included as acomparison. 76% of mice immunised with the AdM42 regime using an eightweek prime-boost interval were completely protected against a lethalchallenge with 10⁴ parasitised red blood cells (pRBCs) as shown in Table1.

TABLE 1 No. Mice Median (Range) Peak % Immunisation Protected/ %Parasitaemia of Regime Challenged Protected Protected Mice AdM42 (2 wks)0/6 0% N/A AdM42 (8 wks) 4/5 + 4/6 + 76%  1.2% (0.004%-27.7%) 5/6 MM42(8 wks) 0/3 0% N/A AdAd42 (8 wks) 0/3 0% N/A Naïve 0/4 + 0/4 + 0% N/A0/4

The table outlines the results from individual experiments and theoverall level of protective efficacy. The median and range of peakparasitaemia of those mice that survived in each group are included.Exponential parasite growth results in ≧80% blood-stage parasitaemiawithin 5-7 days post-infection in naïve or unprotected mice, at whichpoint mice are sacrificed. Protected mice can control and ultimatelyclear blood-stage malaria infection.

These results could be replicated in a second strain of mouse, and inthis case 100% of C57BL/6 mice survived challenge, compared to none ofthe naïve unimmunised controls as shown in Table 2.

TABLE 2 No. Mice Median (Range) Peak Immunisation Protected/ % %Parasitaemia of Regime Challenged Protected Protected Mice AdM42 (8 wks)6/6 100% 14.7% (3.7%-56.4%) Naïve 0/6  0% N/A

100% of BALB/c mice immunised with this regime were also protectedagainst a challenge with 50 P. yoelii sporozoites—the natural mode ofmalaria infection (FIG. 8).

1.5 Sterile Protection of Mice to P. Berghei Sporozoite Challenge byAdHu5-PbCSP Immunisation.

AdHu5 vector recombinant for the circumsporozoite protein (CSP) from P.berghei was generated, with the antigen under the control of the longpromoter (33). BALB/c mice were immunised as indicated in Table 3. Somegroups of mice received a single immunisation i.d. of AdHu5 expressingPbCSP and were challenged two or eight weeks later. The remaining groupswere immunised with heterologous prime-boost regimes using AdHu5 and MVAexpressing PbCSP. The time interval in weeks between the twoimmunisations is indicated in parentheses. All immunisation regimesutilised the AdHu5 vector expressing PbCSP under the control of the longpromoter. Mice were challenged i.v. with 10³ P. berghei sporozoites.Blood-stage parasitaemia was monitored daily by Giemsa-stainedthin-blood smear from day 5 in challenged mice. Mice are protected giventhe continued absence of patent blood-stage parasitaemia up until day21. 33% of mice were protected against challenge following a singleimmunisation with Ad-PbCSP and infection two weeks later as shown inTable 3.

TABLE 3 Time Interval between No. Mice Immunisation Immunisation andProtected/ % Regime Challenge Challenged Protection Ad-PbCSP 2 weeks 4/12 33% Ad-PbCSP 8 weeks 1/6 18% Naïve 2 weeks/8 weeks  0/12  0%Ad-MVA 2 weeks 66% PbCSP (2 week prime- boost interval) Ad-MVA 2 weeks100%  PbCSP (8 week prime boost interval) Naïve 2 weeks  0%

Immunisation with Ad-PbCSP induces a potent CDS⁺ T cell response againstthe H-2^(d) class I-restricted epitope, Pb9 (34). If these mice areboosted with MVA encoding PbCSP eight weeks later, then 100% of mice arerefractory to P. berghei sporozoite challenge Table 3 and Ref. (33).

Example 2 Production of Pre-Erythrocytic Human Malaria (P. Falciparum)Antigen Vaccines with Human and Simian Adenovirus Vectors

Vaccination with pre-erythrocytic vaccines have shown particular promisefor tacking the huge global health problem of malaria (1,2) with someefficacy in clinical trials from immunity to this stage of the malarialife cycle directed towards the sporozoite and subsequent intrahepaticschizont (37). The cellular immune response has previously been shown tobe important in pre-erythrocytic immunity with CD8⁺ T cells andIFN-gamma production playing a central role in protection to liver stagemalaria (38). The thrombospondin-related adhesion protein (TRAP) is anantigen expressed on the sporozoites which has previously been shown toinduce a protective CD8⁺ T cell responses (39). TRAP has beenextensively tested in vaccine clinical trials as a fusion protein with amultiepitope string containing additional B-cell, CD8⁺ and CD4⁺ T cellepitopes, known as ME.TRAP (40,41). In humans, FP9-MVA.ME.TRAPprime-boost regimes have been shown to induce CD8⁺ as well as CD4⁺ Tcell responses that conferred sterile protection in some volunteers(42,43). Adenoviral vectors of the human serotype 5 have previously beenused in a P. yoelii mouse model of malaria and have shown outstandingimmunogenicity and significant protection after just a single dose (44).However, one major limitation preventing the use of this serotype inhumans is the ubiquitous presence of AdH5, with frequent childhoodinfections resulting in seroconversion. It has been reported that nearlyall adults have antibodies against AdH5 (45), and 45% to 80% ofindividuals possess neutralizing antibodies (NAB) to the virus (46). Tocircumvent the problem of preexisting immunity to AdH5, there has beenincreased interest in the use of adenoviral serotypes of simian originthat do not circulate at appreciable levels in human populations, with anumber of studies demonstrating the ability of these vectors to elicitCD8⁺ T-cell responses in both mice and nonhuman primate models of SARS(47) and HIV (48, 49).

In this current work, the inventors demonstrate for the first time in amouse malaria model that with the use of a long intron A containing CMVpromoter, as defined above, four simian adenoviral vectors, AdC6, AdC7,AdC9 (also known as C68 (50)), can induce outstanding CD8⁺ T cellresponses often outperforming AdH5. Moreover, there was induction ofhigh levels of sterile protection to a challenge with P. berghei after asingle vaccination with the vectors. Finally, in conditions ofpreexisting immunity to AdH5 simian adenoviral vectors still maintaineda high degree of protection which was abrogated with the use of humanserotype 5.

2.1 Material and Methods

Mice and Immunizations

Female BALB/c mice 4 to 6 week of age were used and immunizedintradermally, which has previously been shown to elicit betterimmunogenicity when compared to other routes e.g. sub-cutaneous,intramuscular (58). MVA.ME.TRAP (MVA) or FP9.ME.TRAP were administeredat a dose of 1×10⁶ or 1×10⁷ pfu, and adenoviruses at a dose of 1×10⁹ or1×10¹⁰ viral particles (v.p.).

Viral Vectors

All vectors express the transgene ME.TRAP that has been previouslydescribed (40,71). The insert ME.TRAP is a hybrid transgene of 2398 byencoding a protein of 789 aa. The ME string contains the BALB/c H-2K^(d)epitope Pb9 amongst a number of other B- and T-cell epitopes (72). Thesimian adenoviral vectors (SAdV) and the AdHu5 vector were constructedwith a intron A bearing long CMV promoted as described (73).Construction of the MVA (71) and FP9 (43) has been described earlier.

Ex Vivo IFNγ ELISPOT

ACK-treated splenocytes or PBMCs were cultured for 18-20 hours onIPVH-membrane plates (Millipore) with the immunodominantH-2K^(d)-restricted epitope Pb9 (SYIPSAEKI) at a final concentration of1 μg/ml. ELISPOT was performed as previously described (74). To analyzethe breadth of the immune response, splenocytes were stimulated withpools of 20-mer peptides overlapping by 10 aa spanning the entire lengthof TRAP (43,71) as well as a pool of peptides covering the ME string,all at a final concentration of 5 μg/ml.

Intracellular Cytokine Staining

ACK-treated splenocytes were incubated for 5 hours in presence of 1μg/ml Pb9 and 4 μl/ml Golgi-Plug (BD). Intracellular cytokine staining(ICS) was performed with BD cytofix/cytoperm plus kit according to themanufacturer's instructions. Splenocytes were stained with a suitablecombination of fluorochrome-conjugated antibodies, specific for CD8(clone 53-6.7, eBioscience), IFNγ (clone XMG1.2, eBioscience), CD27(clone LG.7F9, eBioscience), CD43 (clone 1B11, BD/Pharmingen), CD127(clone A7R34, eBioscience), IL-2 (clone JES6-5H4, eBioscience), mouseisotype controls IGg2a (eBR2a, eBioscience), CD16/CD32 Fcgamma III/IIReceptor (2.4G2, BD/Pharmingen), anti-Granzyme B (clone GB12, Caltag),IgG1 isotype control (Caltag). When CD62L (clone MEL-14, eBioscience)was used, stimulated cells were incubated with TAPI-2 peptide (PeptidesInternational, USA) at a final concentration of 250 μM to prevent CD62Lshedding from the cell surface. For peptide mapping and potency inC57BL/6 mice, splenocytes were stimulated with peptide pools containing20-mers overlapping by 10, spanning all of the ME-TRAP sequence. Thefinal concentration was 20 μg/ml. CD4 and CD8 responses were tracked byflow cytometry and individual peptides were synthesized after ananalysis in the SYFPEITHI database to predict the immunodominantepitopes. Upon titration, individual peptides were used at a finalconcentration of 5 μg/ml.

Flow cytometric analyses were performed using a FACSCanto (BDBiosciences) and data were analyzed with either FACSDiva (BD) or Flow Jo(Tree Star) software.

Evaluation of Antigen-Specific CD8⁺ T-Cell Response by Flow Cytometry

The frequency of IFNγ⁺ CD8⁺ T cells was calculated by subtracting thevalues from the unstimulated control, which never exceeded 0.1% in anyof the experiments. The total number of antigen-specific cells wascalculated as previously described (53). For the phenotypic makersinvestigated, each marker was compared to an isotype control.

ELISA

IgG antibodies against the TRAP region were analyzed by ELISA asdescribed previously (43). For this experiment, serum was obtained fromgroups of at least 3 BALB/c mice after 2 weeks of immunization withindividual vectors. Results were reported as a dilution factor neededfor a sample in order to reach the O.D. of a naïve serum.

Parasite Challenge

Plasmodium berghei (ANKA strain clone 234) sporozoites (spz) wereisolated from salivary glands of female Anopheles stephensi mosquitoes.Parasites were resuspended in RPMI-1640 medium with each mouse receivinga total of 1,000 spz via the i.v. route. Blood samples were taken ondaily basis from day 5 to 20; smears were stained with Giemsa andscreened for the presence of schizonts within the red blood cells.Survival was defined as complete absence of parasites in blood.

2.2 Results

Breadth of the Immune Response.

The breadth of the immune response to ME.TRAP was analyzed in BALB/c(FIG. 9 a) and in C57BL/6 (FIG. 10 a) mice by IFNγ ELISPOT. In BALB/c,the predominant response was directed towards the immunodominantH-2K^(d)-restricted epitope Pb9, whereas in C57BL/6 the response waspresent in three sub-pools: the ME string, TRAP 1 and TRAP 2. Additionalanalysis with intracellular cytokine staining and the use of SYFPEITHIdatabase allowed the characterization of a CD4 epitope in TRAP 1(IHLYVNVFSNNAKEI), a CD8 epitope in TRAP 2 (NVAFNRFLV) and a CD8 epitopein the ME string (DASKNKEKAL).

Kinetics of the Pb9-Specific CD8⁺ T Cell Response.

The CD8⁺ T cell response to Pb9 from all six vectors was investigated interms of expansion, contraction and generation of memory cells. Thesimian adenovirus (SAds) AdC7 (C7), AdC9 (C9) elicited the strongestimmune responses, followed by AdH5 (H5) (FIG. 9 b, 9 c, 9 d). Of theSAds, AdC6 (C6) was the least potent in terms of IFN-γ production butall Ads induced similar CD8⁺ T-cell expansion kinetics with a peakresponse about 20 days post-immunization. On the other hand, thepoxviruses MVA and FP9 (which use a non-CMV poxvirus promoter) inducedan immune response that peaked one week post vaccination, with adecrease in the frequency of CD8IFN-γ⁺ cells observed as early as the 2weeks post vaccination. The frequency of IFNγ⁺ CD8⁺ T cells at day 60post-vaccination was highest in mice that were vaccinated with anadenovirus, this was most apparent in mice immunized with either C9 orH5. Thus, all adenoviral vectors share similar characteristics in termsof both strength and kinetics of the CD8⁺ T cell response, with profounddifferences in the expansion and contraction kinetics observed betweenadenoviral and poxviral vectors.

Effector CD8⁺ T-Cell Response.

Due to the short interval between infection and progression to disease,effector CD8⁺ T cells can play an important role in protection againstmalaria. Therefore, the acquisition of effector functions determined bythe expression of a number of phenotypic markers, CD43 and Granzyme B,was investigated. CD43 expression has previously been shown to beupregulated during the effector phase of the CD8 response (51) while thecytolytic effector molecule Granzyme B (GrB), is highly expressed inCD8⁺ T effector cells, with lower levels observed in T_(EN) and T_(CM)(52), and it is one of the main mechanisms that CTLs use to killinfected cells. In general, adenoviral vectors induced a significantlyhigher percentage of CD8⁺IFN-gamma⁺CD43^(hi) over the entire course ofthe immune response when compared to the poxviral vectors.Interestingly, poxviral vectors induced a low percentage of CD43^(hi) asearly as one week post-vaccination, suggesting a more rapid transitiontowards the memory phase especially with MVA. At day 60 post-prime, miceimmunized with the adenoviral vectors still retained a significantlyhigher percentage of CD43^(hi) when compared to the poxviral counterpart(FIG. 11 a). In addition, levels of GrB were significantly lower in theMVA group at day 20 (p<0.001), and at day 60 both poxviral vectors weresignificantly lower than C6 (p<0.05) (FIG. 11 b, 11 c). These resultsdemonstrate that in response to all four adenoviruses there was a fulldevelopment of an effector response with preservation of cytolyticmolecules for long periods of time, indicative of the presence of T_(EM)cells (52).

Functional and Phenotypic Memory Markers of Pb9-Specific CD8⁺ T Cells.

One of the main objectives of any vaccination regime is the generationof memory CD8⁺ T cells that are capable of persisting in vivo andexpanding rapidly upon encounter with pathogens thus affordingprotection. To date a number of different molecules have been suggestedto correspond to different sub-types of memory cells (53, 54). In thiscurrent study the inventors chose to investigate a number of thesemolecules to determine whether individual vectors induced differentmemory cells populations. During the early phase of the response, CD8⁺IFN-γ⁺ cells generated in response to either FP9 or MVA displayed aCD62L^(hi), CD127^(hi) and produced IL-2, confirming the rapidtransition towards a T_(CM) phenotype. Conversely, the adenoviralvectors did not induce a central memory CD8⁺ T phenotype, even by day60, with the majority of CD8⁺IFN-γ⁺ cells displaying predominantly aneffector memory phenotype (CD62L^(lo), CD127^(hi), and low percentage ofIL-2 producing cells) (FIG. 12 a-d). Interestingly, CD27 remained low inmice vaccinated with adenoviral vectors, whereas the percentage of CD27⁺cells increased over time in response to the poxviral vectors. SinceCD8⁺ CD27⁻ cells are maintained in response to persistent antigenicstimulation (55), this may suggest that prolonged antigen stimulationwas occurring in response to the adenoviruses. In summary, these resultsdemonstrate that vaccination with adenoviral vectors inducespredominantly a T_(EM) response, as evidenced by a CD62L^(lo), CD127⁺,IL-2^(low) phenotype in addition to the relative high percentage ofCD43^(hi) cells as well as higher levels of cytolytic molecules 60 dayspost-prime.

Survival Following a Challenge with P. Berghei.

To assess the level of protection afforded by these different vectors,mice were challenged with P. berghei as shown in Table 1. BALB/c micewere immunized with adenoviral (1×10¹⁰ vp) and poxviral vectors (1×10⁷pfu) and then challenged 14 days (n=12) and 60 days later (n=6) by i.v.administration of 1000 sporozoites of Plasmodium berghei. Preexistingimmunity to AdH5 was analyzed after injecting groups of 6 BALB/c micewith 5×10⁵ v.p. of AdH5 coding for an unrelated transgene (Ag85.A). 30days later, the same mice were immunized with 1×10¹⁰ v.p. per mouse ofAdH5, C6, C7 and C9 coding for ME.TRAP. Mice were challenged 14 daysafter the last immunization. Numbers represent the percentage of animalsthat survived the challenge. Statistical differences are indicated as: *p<0.05, ** p<0.01, *** p<0.001, and show comparison of individualregimes with the naïve control.

TABLE 1 Pre-existing immunity Day 14 Day 60 to H5 (day 14) (n = 12) (n =6) (n = 6) Vector % % % H5 83 ***  0  0 C6 67 **  0 17 C7 83 *** 50 * 33** C9 92 *** 17 50 * MVA  0  0 n.t. FP9  0  0 n.t. Naive  0  0  0

In conditions with no previous immunity to AdH5 (day 14), C9 providedthe best protection (92%), this was followed by C7, H5 (83%) and finallyC6 (67%), all of them significantly higher than the naïve control group(0%, p<0.001). No protection was afforded by MVA or FP9 at the same timepoint. At day 60 post-prime, significant protection was achieved with C7(50%) and C9 (17%) but no protection was observed when mice wereimmunised with either H5 or C6. The ability of these vectors to conferprotection in presence of preexisting immunity to AdH5, which wouldmimic a human situation where at least 45% of the population is expectedto have NABs to AdH5, was also assessed in this study. Mice wereinitially immunised with AdH5 containing an unrelated insert (Antigen85A from Mycobacterium tuberculosis) which was followed 4 weeks later bythe Ads vectors coding for ME.TRAP. In the presence of pre-existing AdH5immunity, immunization with H5 gave no protection while C9 gave the bestprotection (50%), this was followed by C7 (33%) and C6 (17%). Both, C9and C7 were significantly higher than H5 and the naïve controls (p<0.05and p<0.01, respectively).

T-Cell Responses to Pf TRAP

To determine that immune responses are elicited by the P. falciparumTRAP within the ME-TRAP transgene, immunogenicity was analyzed byintracellular cytokine staining in splenocytes of C57BL/6 mice uponvaccination with all vectors. The ELISpot technique showed an immuneresponse elicited by three sub-pools (FIG. 10 a). Flow cytometricanalysis revealed the presence of one CD4 and one CD8 epitope in theTRAP sequence and a CD8 epitope in the ME string. Additional analysisusing the SYFPEITHI database allowed the identification of the optimalpeptide sequences for synthesis purposes (56). A similar trend in termsof potency of the CD4⁺- and CD8⁺-T cell responses was observed withrespect to the Pb9 responses. C9 elicited the most potent TRAP T-cellresponses, followed by C7, H5 and finally C6. Both poxviral vectorsinduced a more modest immune response measured on week 3post-vaccination (FIG. 10 b).

Antibodies to TRAP

Induction of a TRAP-specific B-cell response by the vectors was assessedin sera from vaccinated mice. All of the adenoviral vectors were able toinduce high levels of IgG antibodies against the TRAP region, whereasthe poxviral vectors elicited low antibody levels. The strongestresponses were achieved by AdH5 (O.D. x=3930±14), followed by AdC7 (x=3358±256), AdC9 ( x=2862±979), AdC6 ( x=739±452); whereas MVA (x=178±98) and FP9 ( x=126±95) induced minimal levels of antibodies (FIG.13). Values for AdH5, AdC7 and AdC9 were significantly higher than therest of the group.

2.3 Discussion

There is increasing evidence that T-cell responses may be a criticalrequirement for protection against diseases such as malaria, AIDS,tuberculosis and cancer. CD8⁺ T cells have previously been shown to playa central role in protection to the liver stage of malaria infection(57). A number of sub-unit vaccines, in the form of naked DNA and viralvectors, have been shown to induce strong CD8⁺ T cell responses in mice(44), providing protection against malaria.

Adenoviral vectors of the human serotype 5 (AdH5) have been tested inmice as vaccine candidates for a variety of infectious diseases (44, 58,59). These vectors have displayed outstanding CD8⁺ T-cell immunogenicityin a prime-boost regime in combination with poxviral vectors and haveconferred significant protection. However, in the only previous study ofadenoviral vectors in the P. berghei model protection by homologous AdH5immunization was minimal (58). Due to the ubiquitous presence of AdH5, ahigh percentage of humans develop antibodies that render the vaccineineffective. To circumvent this problem, adenoviral vectors have beenengineered from chimpanzee serotypes that do not circulate in humans.Previous studies have shown the ability of the chimpanzee adenovirus toelicit potent B- and CD8⁺ T-cell-mediated immune responses in models ofrabies (50), SARS (47) and HIV (49), as a prime or heterologousprime-boost regimes in mice and primates.

The use of three chimpanzee adenoviral vectors, AdC6, AdC7 and AdC9 as apre-erythrocytic malaria vaccine has been possible using an intron Abearing long promoter. The inventors have compared these vectors to AdH5also expressing a long promoter and two poxviral vectors that have beenwidely used in human clinical trials, MVA and FP9.

The adenoviral vectors elicited the most potent CD8⁺ T cell responses,which peaked at week 3 yet maintained a high frequency of Pb9 specificcells even out to 60 days post-prime. In contrast, poxviral vectorspeaked around the first week and then contracted rapidly. Upon analysisof a number of phenotypic markers, such as CD43 and GrB, Ads were shownto induce a potent effector population of cells which was significantlylower when mice were immunized with either of the poxviral vectors. Inaddition, MVA was shown to induce a lower level of GrB suggesting anoverall reduced level of cytotolytic activity. Thus, adenoviral vectorswere able to induce a sustained CD8⁺ T-cell effector response that wasretained at high levels for at least 60 days after priming. Additionalphenotypic markers showed that poxviral vectors induced the generationof a predominantly CD62L⁺, CD127⁺ CD8⁺ T cells, whereas the predominantphenotype of CD8⁺ T cells in response to the Ad vectors wasCD62L^(dull/−), CD127⁺ over a long period of time. Based on expressionof these markers, three different subsets of Ag-specific CD8⁺ T cellscan be identified: effector T cells T_(E) (CD62L⁻CD127⁻); effectormemory T cells T_(EM) (CD62L⁻CD127⁺) and central memory T cells T_(CM)(CD62L⁺CD127⁺) (60).

Antibodies to the TRAP region were also assessed after vaccination ofBALB/c mice with each vector. Potency in terms of the B-cell responsecorrelated well with the magnitude of CD8⁺ T cell responses. Protectionagainst P. berghei in this system relies on CD8⁺ T-cell responsesdirected towards an immunodominant epitope, Pb9. Antibodies to TRAPwould not play a role in protection due to the fact that the TRAPsequence is derived from P. falciparum. However, the presence ofantibodies could add an extra benefit to improve protection in humaninfections with P. falciparum.

The inventors show that the simian adenoviral vectors using a longpromoter elicited potent CD8⁺ T cell responses that are important inprotection in a preerythrocytic mouse model of malaria. In contrast torare human adenovirus serotypes, such as AdHu35 (70), the immunogenicityand efficacy of these simian vectors is as great or greater than AdH5.Comparison of the adenoviral vectors to two poxviral vectors, FP9 andMVA, demonstrated that the Ads were able to sustain a high number ofCD8⁺ T cells over a long period of time that subsequently resulted inthe generation of a high number of T_(EM) cells. Conversely,immunization with either of the poxviral vectors induced a highproportion of T_(CM) cells very early after immunization. In addition,all simian adenoviral vectors induced outstanding levels of protectionduring the effector phase of the response in absence and presence ofpreexisting immunity to AdH5, with protection being maintained for along period of time with a number of the vectors. These data demonstratefor the first time that a single dose of a subunit vaccine is able toelicit protection to P. berghei and highlights the potential of thesimian adenoviral vectors for a future application as a malaria vaccinein humans.

Example 3 Blood Stage Vaccines Against P. Falciparum Malaria

Based on the findings of Example 1 demonstrating the surprising abilityof a Ad-MVA heterologous prime-boost immunisation regime to inducestrong protective immunity to blood stage malaria in a P. yoelii murinemalaria model, the inventors proceeded to generate adenovirus and MVAvectors encoding sequences from the MSP-1 gene of the human malariaparasite P. falciparum. This gene is dimorphic with two prevalencesequence types. It has a well studied block structure that allows theidentification of conserved and variable blocks.

The PfM117 insert (see SEQ ID NO. 1) has been designed as a usefulinsert for immunisation. It comprises conserved sequence blocks 1, 3, 5and 12 at the N terminus of a fusion protein followed by both copies ofthe important 33 kd fragment and at the C terminus of the protein therelatively conserved 19 Kd fragment. The 33 Kd fragment is a wellstudied immunogenic component of the MSP1 antigen that is known tocontain T cell epitopes. It is however dimorphic with substantialsequence divergence between the two major types, often denoted by thelabels Wellcome and MAD20 referring to the parasite strains that earlysequences were derived from (Miller L. H. et al. Mol Biochem Parasitol.1993, 59(1):1-14). In PfM117 the Wellcome strain sequence is foundN-terminal to the MAD20 strain sequence, and immediately C-terminal tothese is the 19 Kd sequence. This latter fragment is highly but notcompletely conserved amongst P. falciparum parasite strains and is knownto be the target of protective antibodies. However some of theseprotective antibodies can be inhibited in their protective action by socalled blocking antibodies (Uthaipaibul et al. J Mol Biol. 2001, 307 (5):1381-94.). Uthaipaibul et al. (2001) describe amino acid changes thatcan be made in the canonical sequence of the 19 Kd fragment that allowinhibitory antibodies to act preferentially over blocking antibodies,thereby increasing the likelihood that antibodies induced to thisfragment should be protective. Therfore within this 19 Kd fragment threeamino acids have been altered to avoid blocking antibody binding(Uthaipaibul et al. (2001)).

The PfM128 insert (see SEQ ID NO. 3), is identical to the PfM117sequence with an additional copy of a 19 Kd fragment inserted betweenthe two 33 Kd fragments. This additional 19 Kd fragment allows analternative allelic sequence of that fragment to be expressed by thisconstruct potentially broadening the range of protective antibodies or Tcells that might be induced by PfM128 compared to PfM117.

We also constructed an additional insert “PfM115” that is very similarto the PfM117 sequence in that it comprises conserved sequence blocks 1,3, 5 and 12 at the N terminus of the PfMSP1 fusion protein followed byboth copies of the important 33 kd fragment and at the C terminus of theprotein the relatively conserved 19 Kd fragment. However there are someminor sequences differences at the end of the 33 Kd fragments comparedto the PfM117 corrected sequence. The PfM115 sequence was used togenerate recombinant vectors of the C6, C7, C9 and AdHu5 serotypes(strain notation as in example 2) using again the intron A containinglong promoter. Good vector genetic stability was observed. Potency asmeasured by antibody induction (FIG. 14) was excellent. In addition bothCD4 and CD8 T cell responses were induced to peptide pools comprisingthe entire insert sequence of PfM115. Boosting with an MVA vectorencoding the same insert led to enhanced antibody and T cell responses.

A widely used in vitro assay to predict the likely efficacy of bloodstage vaccines against P. falciparum is the Growth Inhibitory Activity(GIA) assay (Bergmann-Leitner et al Am J Trop Med Hyg. 2006, 75:437-42;Malkin et al. Infect Immun. 2005, 73:3677-85.) This in vitro assayquantifies the % growth inhibition of blood-stage P. falciparum malariaparasites when cultured in the presence of test and control serum. Aparasite enzymatic reaction is used to quantify parasite growthfollowing the 40 hour time period of the assay. It is hoped that animalsimmunised with candidate blood-stage malaria vaccines will developprotective antibody responses. Serum from these animals can thus bescreened using this assay for their ability to inhibit parasite growth.Considerable efforts have been made to standardise this assay,particularly by the NIH laboratory of C. Long. Mice were immunised withthe AdHu5-PfM115 insert and boosted with the corresponding MVAconstruct. Sera taken at a terminal bleed showed 46-52% GIA, as measuredby the C. Long lab, a level that represents substantial inhibition ofthe growth of blood stage parasites (Bergmann-Leitner et al Am J TropMed Hyg. 2006, 75:437-42). This result suggests that a correspondingimmunisation regime used in humans will show protective efficacy.

To try to increase further the immunogenicity and likely protectiveefficacy of adenoviral vectored vaccines the AdHu5-PfM115 vectoredvaccines was coadminstered as a mixture with the CpG sequence 1826(Brunner et al. J Immunol. 2000, 165:6278-86). CpGs have been wellstudied as adjuvants for protein-based but not for vectored vaccines(Daubenberger C A, Current Opinion in Molecular Therapy 2007; 9:45-52).One previous study of a CpG sequence co-administered with an adenovirusvaccine encoding a tumour antigen PSA led to lower T cell immunogenicitythan when administered without the CpG (Lubaroff et al. Vaccine 2006,24:6155-62). However, coadministration of the 1826 CpG oligonucleotidewith the AdHu5-PfM115 vaccine led to increased CD4 and CD8 T cellresponses as measured by flow cytometry (FIG. 15). This suggests thatcoadministration of CpG oligonucleotides with certain viral vectors,including heterologous long promoter adenoviral vectors, may lead toenhanced T cell immunogenicity for a variety of antigenic inserts.

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1. An adenoviral vector comprising a promoter and a nucleic acidsequence encoding a pathogen or tumour antigen under the control of saidpromoter, wherein said promoter comprises the regulatory element andenhancer of the CMV IE1 promoter an a fragment of the 5′ untranslatedregion of the CMV IE1 gene including intron A, and wherein said antigenis not a murine malaria parasite antigen.
 2. The adenoviral vectoraccording to claim 1, wherein the adenoviral vector is a simianadenoviral vector.
 3. The adenoviral vector according to claim 1,wherein the simian adenoviral vector is AdC6 (C6), AdC7 (C7), or AdC9(C9) vector.
 4. The adenoviral vector according to claim 1 wherein thepromoter excludes Exon B.
 5. (canceled)
 6. The adenoviral vectoraccording to claim 1, wherein the antigen is from a pathogen whichcauses malaria.
 7. The adenoviral vector according to claim 6, whereinthe antigen is a P. falciparum antigen.
 8. The adenoviral vectoraccording to claim 6, wherein the malaria antigen is a pre-erythrocyticor blood-stage malaria antigen.
 9. The adenoviral vector according toclaim 1, wherein the antigen is a malaria antigen selected from thegroup consisting of ME-TRAP, CSP, MSP-I or fragments thereof, and AMAl.10. The adenoviral vector according to claim 9, wherein the malariaantigen is an MSP-I antigen has the sequence of PfM117 (SEQ ID NO. 1) orPfM128 (SEQ ID NO. 3). 11-13. (canceled)
 14. An immunogenic compositioncomprising the adenoviral vector of claim 1 admixed with one or morepharmaceutically acceptable vehicles, carriers, diluents, or adjuvants.15-32. (canceled)
 33. A product, combination or kit comprising; a) apriming composition comprising an adenoviral vector, comprising apromoter and a nucleic acid sequence encoding a pathogen or tumourantigen under the control of said promoter, wherein said promotercomprises the regulatory element and enhancer of the CMV IE1 promoterand a fragment of the 5′ untranslated region of the CMV IE1 geneincluding intron A and b) a boosting composition comprising arecombinant pox virus vector, said pox virus vector further comprisingat least one nucleic acid sequence encoding a pathogen or tumour antigenwhich is the same as at least one antigen of the priming composition.34. (canceled)
 35. The product, combination or kit according to claim33, wherein the promoter excludes Exon B of the CMV IE1 gene. 36.(canceled)
 37. The product, combination or kit according to claim 33,wherein the antigen is from a pathogen which causes malaria.
 38. Theproduct, combination or kit according to claim 37, wherein the antigenis a P. falciparum antigen.
 39. (canceled)
 40. The product, combinationor kit according to claim 33, wherein the antigen is a malaria antigenselected from the group consisting of ME-TRAP, CSP, MSP-I or fragmentsthereof, and AMAl.
 41. The product, combination or kit according toclaim 40, wherein the malaria antigen is an MSP-I antigen having thesequence of PfM117 (SEQ ID NO. 1) or PfM128 (SEQ ID NO. 3). 42-44.(canceled)
 45. A method of eliciting an immune response in a subjectcomprising administering to the subject an effective amount of anadenoviral vector according to claim 1 sufficient to elicit an immuneresponse.
 46. A method of eliciting an immune response in a subjectcomprising administering to the subject an effective amount of theproduct, combination or kit of claim
 33. 47. The method of claim 46,wherein the boosting composition is administered 2-8 weeks after thepriming composition. 48-58. (canceled)
 59. A method for enhancing the Tcell immunogenicity of an immunogenic composition according to claim 14,comprising administering said immunogenic composition in combinationwith a CpG adjuvant.
 60. The method according to claim 59, wherein theCpG adjuvant is administered prior to, concomitantly with, orsubsequently to said immunogenic composition.
 61. A compositioncomprising an immunogenic composition according to claim 14 and a CpGadjuvant. 62-63. (canceled)
 64. The adenoviral vector according to claim1, wherein the promoter has a length of about 1.9 kb.
 65. The adenoviralvector of claim 1, wherein the promoter has the nucleotide sequenceshown in SEQ ID NO: 7.